Construction of synteny groups of Brassica alboglabra by RAPD markers and detection of chromosome aberrations and distorted transmission under the genetic background of B. campestris

Interspecific hybrids were produced by crosses between the inbred lines of B. campestris and B. alboglabra, and were backcrossed twice to B. campestris. Genetical constitutions of the BC₂ plants were analyzed by RAPD (random amplified polymorphic DNA), flow cytometry and cytological observations. By using 140 arbitrary primers, a total of 137 polymorphic bands were obtained and 125 were found to be specific to B. alboglabra. Based on the presence and absence of the specific RAPD markers of B. alboglabra, 13 synteny groups were constructed. The number of markers in each synteny group was found to be different and varied from 2 to 28. This reflects the difference in the degree of genetic variability among the B. alboglabra chromosomes from those of B. campestris. Losses or gains of RAPD markers were observed frequently in most of the synteny groups, which indicated the occurrence of chromosome translocations and/or deletions in the chromosomes of B. alboglabra. In a population of 40 BC₂ plants, chromosome transmission rates were analyzed by using the RAPD markers in each synteny group. Most of the chromosomes of the synteny groups were transmitted with rates of 0.37–0.68. An extremely high transmission rate, 0.98, was however observed in one of the synteny groups. Inheritance data of the synteny groups revealed relationships among themselves. The plants lacking the RAPD markers of two synteny groups tended to lose others belonging to the rest of the synteny groups, indicating the effects of these groups on the transmission of B. alboglabra chromosomes to the B. campestris background. Received: 26 February 1999 / Accepted: 30 December 1999     

Characterization of disomic addition lines Brassica napus-Brassica nigra by isozyme,fatty acid,and RFLP markers

Summary Six Brassica napus — B. nigra disomic addition lines were characterized by isozyme, fatty acid, and RFLP markers. The markers were arranged in six synteny groups, representing six of the eight chromosomes present in the B. nigra genome. Synteny group 1 displayed high levels of linoleic and linolenic acids in the seeds of the B. nigra parent. Synteny group 3 accumulated higher levels of eicosenoic and erucic acid than B. nigra. Three of the lines transmitted the alien chromosome to 100% of the progeny. The rest had variable transmission rates but all were above 50%. Most of the lines produced disomic addition plants in their progeny, suggesting pollen transmission of the alien chromosome. In addition to the marked lines, six others remained unmarked. These could be grouped into two classes according to their alien chromosome transmission. It is likely that they represent the two other B. nigra chromosomes that remained uncharacterized by the markers. No diploid individuals carrying B. nigra genome-specific markers were detected in the progenies studied.     

Location of genes coding isozyme markers on Aegilops umbellulata chromosomes adds data on homoeology among Triticeae chromosomes

Summary Zymogram analysis was used to identify the Aegilops umbellulata chromosomes that carry the structural genes for particular isozymes. Wheat, Aegilops and wheat-Aegilops hybrid derivative lines (which contained identified Aegilops chromosomes) were tested by gel electrophoresis for isozymes of particular enzymes. It was found that Aegilops chromosome A (nomenclature according to G. Kimber 1967) carries a structural gene for 6-phosphogluconate dehydrogenase, Aegilops chromosome B carries structural genes for glucose phosphate isomerase and phosphoglucose mutase, Aegilops chromosome D carries genes for leaf peroxidases, Aegilops chromosome E carries structural genes for endosperm peroxidases, acid phosphatases and leaf esterases, Aegilops chromosome F carries a gene for embryo plus scutellum peroxidases and Aegilops chromosome G carries structural genes for endosperm alkaline phosphatases, leaf alkaline phosphatases and leaf esterases. The results obtained indicate that chromosome B is partially homoeologous of the wheat chromosomes of group 1 and 4, and chromosome E is partially homoeologous of wheat chromosomes of groups 7 and 4. Circumstantial evidence is also provided about the possible association between chromosomes C, D and A of A. umbellulata respectively with chromosomes 5, 2 and 1 of wheat.     

Asymmetric somatic hybrids of Brassica: partial transfer of B. campestris genome into B. oleracea by cell fusion

Summary To examine the possibility of producing asymmetric somatic hybrids of Brassica having a complete genome of one species and a part of the other, we fused inactivated B. oleracea protoplasts with X-irradiated B. campestris protoplasts. The plants obtained were studied with regard to their morphology, isozymes and chromosomes. The morphology of the hybrids was similar to B. oleracea in 9 out of 22 hybrids studied and the rest showed the intermediate phenotype of the parents. Analysis of three isozymes, leucine aminopeptidase, acid phosphatase and esterase indicated that ten hybrids lost B. campestris-specific bands in one or more of the three isozymes examined. The chromosome analysis showed that 90% of the hybrids were aneuploids. In addition, abnormal chromosomes were often found in root tip cells. These results suggested that the hybrids obtained were asymmetric in nature and resulted from elimination of B. campestris chromosomes by X-ray irradiation.     

A full set of monosomic addition lines in Beta vulgaris from Beta webbiana: morphology and isozyme markers

Summary Nine different monosomic additions in Beta vulgaris from Beta webbiana were characterized through morphological characters and isozyme markers. The effect of the alien chromosome on the morphology of the recipient species is chromosome specific, and nine morphotypes could be distinguished. The added chromosome caused a growth reduction in the recipient plants. Eleven isozyme systems were used as marker systems. A 6PGDH band was found as a marker for chromosome 7, which contains a resistance gene for the beet cyst nematode in monosomic additions from Beta procumbens and Beta webbiana. A difference in the 6PGDH zymogram pattern between the two species with respect to this chromosome has been noted.     

Molecular-mapping analysis in Brassica napus using isozyme, RAPD and RFLP markers on a doubled-haploid progeny

We have undertaken the construction of a Brassica napus genetic map with isozyme (4%), RFLP (26.5%) and RAPD (68%) markers on a 152 lines of a doubled-haploid population. The map covers 1765 cM and comprises 254 markers including three PCR-specific markers and a morphological marker. They are assembled into 19 linkage groups, covering approximatively 71% of the rapeseed genome. Thirty five percent of the studied markers did not segregate according to the expected Mendelian ratio and tended to cluster in eight specific linkage groups. In this paper, the structure of the genetic map is described and the existence of non-Mendelian segregations in linkage analysis as well as the origins of the observed distortions, are discussed. The mapped RFLP loci corresponded to the cDNAs already used to construct B. napus maps. The first results of intraspecific comparative mapping are presented.     

A map of rye chromosome 2R using isozyme and morphological markers

Summary The segregation of isozymic loci for leaf peroxidases (L2Per) has been investigated in backcrosses and F₂ offspring of rye lines having purple seeds (Ps) and monstrosum ears (mo). The Ps, L2Per-3b, mo, and L2Per-2 loci were linked. The Ps and mo loci have been previously located on the 2R chromosome, and the L2Per-3b and L2Per-2 loci have been located on the 2RS chromosome arm. The results favor the gene order Ps ... L2Per-3b ... mo ... L2Per-2 or Ps ... mo... L2Per-3b ... L2Per-2. The position of the loci relative to the centromere is still not known, but the obtained results suggest that the mo locus could be located on the 2RS chromosome arm. On the basis of previously reported linkage groups, the most probable arrangement of the loci located on chromosome 2R is: dw2 ... Ps ... (L2Per-3a ... L2Per-3b ... mo) ... L2Per-2. It has not been possible to know the position of L2Per-4 loci (also located on 2RS chromosome arm) relative to L2Per-3a and L2Per-3b loci.     

A genetic map of Blumeria graminis based on functional genes, avirulence genes, and molecular markers

A genetic map of the powdery mildew fungus, Blumeria graminis f. sp. hordei, an obligate biotrophic pathogen of barley, is presented. The linkage analysis was conducted on 81 segregating haploid progeny isolates from a cross between 2 isolates differing in seven avirulence genes. A total of 359 loci were mapped, comprising 182 amplified fragment length polymorphism markers, 168 restriction fragment length polymorphism markers including 42 LTR-retrotransposon loci and 99 expressed sequence tags (ESTs), all the seven avirulence genes, and a marker closely linked to the mating type gene. The markers are distributed over 34 linkage groups covering a total of 2114 cM. Five avirulence genes were found to be linked and mapped in clusters of three and two, and two were unlinked. The Avr(a6) gene was found to be closely linked to markers suitable for a map-based cloning approach. A linkage between ESTs allowed us to demonstrate examples of synteny between genes in B. graminis and Neurospora crassa.     

Analysis of the Brassica oleracea genome by the generation of B. campestris-oleracea chromosome addition lines: characterization by isozymes and rDNA genes

Summary This study aimed at generating chromosome addition lines and disclosing genome specific markers in Brassica. These stocks will be used to study genome evolution in Brassica oleracea L., B. campestris L. and the derived amphidiploid species B. napus L. B. campestris-oleracea monosomic and disomic chromosome addition plants were generated by crossing and backcrossing the natural amphidiploid B. napus to the diploid parental species B. campestris. The pollen viability of the derived sesquidiploid and hyperploid ranged from 63% to 88%, while the monosomic and disomic addition plants had an average pollen fertility of 94% and 91%, respectively. The addition lines were genetically characterized by genome specific markers. The isozymes for 6PGD, LAP, PGI and PGM, and rDNA Eco RI restriction fragments were found to possess the desired genome specificity. Duplicated loci for several of these markers were observed in B. campestris and B. oleracea, supporting the hypothesis that these diploid species are actually secondary polyploids. A total of eight monosomic and eight disomic addition plants were identified and characterized on the basis of these markers. Another 51 plants remained uncharacterized due to the lack of additional markers. rDNA genes were found to be distributed in more than one chromosome, differing in its restriction sites. Intergenomic recombination for some of the markers was detected at frequencies between 6% and 20%, revealing the feasibility of intergenomic gene transfer.     

The morphology, cytology, and C-banded karyotypes of Brassica campestris, B. oleracea, and B. napus plants regenerated from protoplasts

The behaviour of Brassica campestris (2n=20, AA), B. oleracea (2n=18, CC), and B. napus (2n=38, AACC) were studied during a tissue-culturing process. Hypocotyl-protoplasts were cultivated into calli from which new plants were regenerated. The regenerated plants were compared, and mitotic root-tip cells were C-banded and karyotyped. A majority of the plants were tetraploid. The meioses were studied in the PMCs. A number of abberations were observed, mainly due to faulty spindle function. There was a difference between the three species in that B. campestris performed the most poorly with many fewer regenerated plants. These plants were more morphologically disturbed and had more problems during pollen production than B. oleracea and B. napus plants.     

A genetic map of melon (Cucumis melo L.) with RFLP, RAPD, isozyme, disease resistance and morphological markers

One hundred and ten markers were analysed for linkage in 218 F₂ plants derived from two divergent cultivars (Védrantais and Songwhan Charmi) of Cucumis melo (L.). Thirty-four RFLPs, 64 RAPDs, one isozyme, four disease resistance markers and one morphological marker were used to construct a genetic map spanning 14 linkage groups covering 1390 cM of the melon genome. RAPD and RFLP markers detected similar polymorphism levels. RFLPs were largely due to base substitutions rather than insertion/deletions. Twelve percent of markers showed distorted segregation. Phenotypic markers consisted of two resistance genes against Fusarium wilt (Fom-1 and Fom-2), one gene (nsv) controlling the resistance to melon necrotic spot virus, one gene (Vat) conferring resistance to Aphis gossypii, and a recessive gene for carpel numbers (3 vs 5 carpels: p).     

Revision and reclassification of three Plasmopara species based on morphological and molecular phylogenetic data

Based on the results of morphological and DNA sequence (partial D1–D3/D7–D8 nuLSU and partial nuSSU-ITS1-5.8S rDNA) data, three species of Plasmopara are revised and reclassified. A species of Plasmopara parasitic on Scorzonera, invalidly published several times, is assigned to a new genus and species under Novotelnova scorzonerae. Plasmopara euphrasiae sp. nov. is segregated from P. densa, and P. centaureae-mollis is revised and relegated to synonymy of Bremia centaureae. All taxa are described and illustrated.     

Formation of nodular structures on the non-legumes Brassica napus,B. campestris,B. juncea and Arabidopsis thaliana with Bradyrhizobium and Rhizobium isolated from Parasponia spp. or legumes grown in tropical soils

Strains of Bradyrhizobium formed nodule-like structures on Arabidopsis and species of Brassica in pots with sandvermiculite and in glass tubes on a nitrogen-free mineral salts agar. Broad-host-range Rhizobium strains NGR234 from Lablab purpureus and NGR76 from Phaseolus vulgaris formed similar nodule-like structures on Brassica spp. The size of these structures on plants in pots were large, often reaching 10 mm in diameter.The frequency of inoculated Brassica plants in pots with nodule-like structures was 25–50%, depending on the inoculum strain. The inheritable nature of factors involved in the formation of the nodule-like structures was demonstrated when the structures occurred on 100% of inoculated B. napus seedlings derived from plants with the nodule-like structures.Nodule-like structures occurred without, but not with, the application of a cellulase-pectolyase-PEG treatment to the roots. Attempts to isolate Bradyrhizobium or Rhizobium from the nodule-like structures failed. Internal infection of these structures could not be detected using either the light or electron microscope. The inoculum strains of root-nodule bacteria were detected in high numbers in the rhizosphere of plants 5 months after inoculation. On agar plates bacterial colonies could be seen, with undiminished growth, over the surface of the agar extending to the root surface. However, ground root tissue of Brassica was toxic to Bradyrhizobium strains. This suggested that Bradyrhizobium strains would not survive after infecting the roots of Brassica spp. Nitrogen fixation was associated with high rhizosphere populations of Azospirillum and not with Bradyrhizobium induced nodule structures of Brassica spp.     

Structure and organization of the B genome based on a linkage map in Brassica nigra

We constructed a genetic map on Brassica nigra based on a segregating population of 83 F₂ individuals. Three different types of molecular markers were used to build the map including isozymes, restriction fragment length polymorphisms (RFLP), and random amplified polymorphic DNA (RAPD). The final map contained 124 markers distributed in 11 linkage groups. The map covered a total distance of 677 cM with the markers distributed within a mean distance of 5.5cM. Of the sequences found in the B. nigra map, 40% were duplicated and organized into three different types of arrangements. They were either scattered throughout the genome, organized in tandem, or organized in blocks of duplicated loci conserved in more than 1 linkage group.     

Genetic characterization of apospory in tetraploid Paspalum notatum based on the identification of linked molecular markers

Tetraploid Paspalum notatum (bahiagrass) is a valuable forage grass with aposporous apomictic reproduction. In a previous study, we showed that apospory in bahiagrass is under the control of a single dominant gene with a distorted segregation ratio. The objective of this work was to identify molecular markers linked to apospory in tetraploid P. notatum and establish a preliminary syntenic relationship with the genomic region associated with apospory in P. simplex. A F₁ population of 290 individuals, segregating for apospory, was generated after crossing a completely sexual plant (Q4188) with a natural aposporous apomictic plant (Q4117). The whole progeny was classified as sexual or aposporous by embryo sacs analysis. A bulked segregant analysis was carried out to identify molecular markers co-segregating with apospory. Four hundred RAPD primers, 30 AFLP primers combinations and 85 RFLP clones were screened using DNA from both parental genotypes and aposporous and sexual bulks. Linkage analysis was performed with cytological and genetic information from the complete progeny. Cytoembryological analysis showed 219 sexual and 71 aposporous F₁ individuals. Seven different molecular markers (2 RAPD, 4 AFLP and 1 RFLP) were found to be completely linked to apospory. The RFLP probe C1069, mapping to the telomeric region of the long arm of rice chromosome 12, was one of the molecular markers completely linked to apospory in P. notatum. This marker had been previously associated with apospory in P. simplex. A preliminary map of the chromosome region carrying the apospory locus was constructed.     

Direct isolation of cDNA sequences from specific chromosomal regions of the tomato genome by the differential display technique

The differential display technique was originally developed for the isolation of differentially expressed genes from eukaryotic tissues. We have adapted this technique for the isolation of cDNA markers from specific regions of the tomato genome. For this purpose, differential display was performed on RNA extracted from leaf tissue of nearly isogenic lines for the Tm-2a gene of tomato. On average, one out of 20 primer combinations resulted in a polymorphism at the cDNA level. When used as hybridization probes, all of these cDNA fragments were single or low copy and all of them were polymorphic on Southern hybridizations using DNA from the isogenic lines. Genetic mapping revealed in each case at least one locus in the introgressed segment on chromosome 9 of tomato. Thus, this technique might provide a way for the direct isolation of transcribed sequences from specific regions of any animal or plant genome for which such lines exist.     

A set of simple PCR markers converted from sequence specific RFLP markers on tomato chromosomes 9 to 12

A set of 24 simple PCR markers was generated for tomato chromosomes 9, 10, 11 and 12. Polymorphism was sought for between Lycopersicon esculentum and one of six other Lycopersicon species (L. parviflorum, L. cheesmanii, L. hirsutum, L. pennellii, L. peruvianum, and L. chilense). PCR primers, which were designed from mapped RFLP sequences, were used to amplify genomic DNA of the different species and the PCR amplification products were screened for polymorphism by testing restriction enzymes. With this approach, 24 (71%) of the 34 selected RFLPs were converted into simple PCR markers. By using a reference population, the map positions of these markers relative to the original RFLP markers were verified. These markers are locus specific and can be efficiently used for alignment of linkage maps, mapping target genes and marker assisted selection.     

A new family of dispersed repeats from Brassica nigra: characterization and localization

The 459-bp HindIII (pBN-4) and the 1732-bp Eco RI (pBNE8) fragments from the Brassica nigra genome were cloned and shown to be members of a dispersed repeat family. Of the three major diploid Brassica species, the repeat pBN-4 was found to be highly specific for the B. nigra genome. The family also hybridized to Sinapis arvensis showing that B. nigra had a closer relationship with the S. arvensis genome than with B. oleracea or B. campestris. The clone pBNE8 showed homology to a number of tRNA species indicating that this family of repeats may have originated from a tRNA sequence. The species-specific 459-bp repeat pBN-4 was localized on the B. nigra chromosomes using monosomic addition lines. In addition to the localization of pBN-4, the chromosomal distribution of two other species-specific repeats, pBN34 and pBNBH35 (reported earlier), was studied. The dispersed repeats pBN-4 and pBNBH35 were found to be present on all of the chromosomes, whereas the tandem repeat pBN34 was localized on two chromosomes.     

Production and genetic analysis of partial hybrids in intertribal crosses between Brassica species (B. rapa, B. napus) and Capsella bursa-pastoris

Capsella bursa-pastoris (L.) Medic (2n = 4x = 32) is a natural double-low (erucic acid < 1%, glucosinolates < 30 micromol/g) germplasm and shows high degree of resistance to Sclerotinia sclerotiorum. Hybridizations were carried out between two Brassica species viz. B. rapa (2n = 20) and B. napus (2n = 38) as female and C. bursa-pastoris as male parent to introduce these desirable traits into cultivated Brassica species. Majority of F(1) plants resembled female parents in morphology and only a few expressed some characters of male parent, including the white petals. Based on cytological observation of somatic cells, the F(1) plants were classified into five types: two types from the cross with B. rapa, type I had 2n = 27-29; type II had 2n = 20; three types from the crosses with B. napus, type III was haploids with 2n = 19; type IV had 2n = 29; type V had 2n = 38. One to two chromosomes of C. bursa-pastoris were detected in pollen mother cells (PMCs) of type I plant by genomic in situ hybridization (GISH), together with chromosomal segments in ovary cells and PMCs of some F1 plants. Amplified fragment length polymorphism (AFLP) bands specific for the male parent, novel for two parents and absent bands in Brassica parents were generated in majority of F1 plants, even in Brassica-types and haploids, indicating the introgressions at various levels from C. bursa-pastoris and genomic alterations following hybridization. Some Brassica-type progeny plants had reduced contents of erucic acid and glucosinolates associated with improved resistance to S. sclerotiorum. The cytological and molecular mechanisms behind these results are discussed.